title: Human-induced pluripotent stem cell-derived cardiomyocytes for studies of cardiac ion transporters.
abstract: Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (iCell Cardiomyocytes) with ion channel activities that are remarkably similar to adult cardiomyocytes. Here, we extend this characterization to cardiac ion transporters. Additionally, we document facile molecular biological manipulation of iCell Cardiomyocytes to overexpress and knockdown transporters and regulatory proteins. Na/Ca exchange (NCX1) and Na/K pump currents were recorded via patch clamp, and Na/H and Cl/OH exchanges were recorded via oscillating proton-selective microelectrodes during patch clamp. Flux densities of all transport systems are similar to those of nonrodent adult cardiomyocytes. NCX1 protein and NCX1 currents decline after NCX1 small interfering (si)RNA transfection with similar time courses (τ ≈ 2 days), and an NCX1-Halo fusion protein is internalized after its extracellular labeling by AlexaFluor488 Ligand with a similar time course. Loss of the cardiac regulatory protein phospholemman (PLM) occurs over a longer time course (τ ≈ 60 h) after PLM small interfering RNA transfection. Similar to multiple previous reports for adult cardiomyocytes, Na/K pump currents in iCell Cardiomyocytes are not enhanced by activating cAMP production with either maximal or submaximal cytoplasmic Na and using either forskolin or isoproterenol to activate adenylate cyclases. Finally, we describe Ca influx-dependent changes of iCell Cardiomyocyte capacitance (Cm). Large increases of Cm occur during Ca influx via NCX1, thereby documenting large internal membrane reserves that can fuse to the sarcolemma, and subsequent declines of Cm document active endocytic processes. Together, these results document a great potential of iCell Cardiomyocytes for both short- and long-term studies of cardiac ion transporters and their regulation.
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical School, Dallas, Texas, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Fine
authors.forename: Fang-Min
authors.initials: FM
authors.lastname: Lu
authors.forename: Mei-Jung
authors.initials: MJ
authors.lastname: Lin
authors.forename: Orson
authors.initials: O
authors.lastname: Moe
authors.forename: Hao-Ran
authors.initials: HR
authors.lastname: Wang
authors.forename: Donald W
authors.initials: DW
authors.lastname: Hilgemann
mesh_terms: D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D004705:Endocytosis; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D017136:Ion Transport; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012508:Sarcolemma / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium
keywords: human-induced pluripotent stem cells; iCell Cardiomyocytes; ion transporters; phospholemman; sodium-calcium exchange; sodium-potassium pumps